Stephen T. Haley writes: > I am trying to do some "relative" quantitative immunoflourescence > on FITC labelled tissue sections using a Bio-Rad MRC-1000. > I was happily collecting data. > The levels of the surface markers I am trying to measure went up or > down as I expected. The trouble arose when I tried to check the > validity of my measurements with some InSpeck beads from Molecular > Probes. The beads are calibrated to several relative intensities. To > make a long story short I found out that the system is not linear. Any chance that spherical aberration is contributing to the non-linearity? I.e. are the test beads at different depths under the coverslip? Another thought: any chance that the beads are photobleaching? Martin Wessendorf PhD, Asst Professor [log in to unmask] Dept Cell Biology and Neuroanatomy (612) 624 2991 (voice) Confocal Microscopy Facility (612) 624 8118 (FAX) University of Minnesota Minneapolis, MN 55455